Studies of the Staphylococcus aureus cid and lrg operons have provided important insight into the regulatory control of bacterial murein hydrolase activity and autolysis and have led to a model in which this system is functionally analogous to the control elements of programmed cell death (PCD) in more complex eukaryotic organisms. Although the Cid and Lrg proteins have been shown to be similar to bacteriophage holins and antiholins, respectively, which are fundamental to the control of cell death and lysis during the lytic stage of a bacteriophage infection, the precise molecular/biochemical mechanisms utilized by their bacterial counterparts during cell death and lysis remain to be determined. In the current proposal, we have built on recent studies in our laboratory demonstrating that cytoplasmic acidification and pyruvate metabolism are critical aspects of bacterial cell death to probe the specific functions of the Cid and Lrg proteins. In the first specific aim we will utilize a molecular genetic approach to examine the relationship between the CidA/B proteins and pyruvate metabolic enzymes in the control of bacterial cell death. The second aim will utilize a biophysical approach to test the model that Cid- and Lrg-mediated transport is a fundamental aspect of the control of bacterial cell death. The third and final aim will explore the CidR-mediated regulation of this system with a focus on the identification of the effector molecule(s) that induces its activity. Overall, the results generated by the experiments described in these aims will illuminate the molecular mechanisms underlying bacterial PCD and uncover the metabolic control elements required for its regulation, ultimately leading to improved therapeutic strategies to fight bacterial infections.